The present invention relates generally to preserving and stabilizing biological materials by freezing, drying, and freeze-drying, and more specifically to protectant mixtures and aqueous preservation media for preserving biological materials, as well as methods of preserving biological materials and the preserved biological material compositions themselves.
The preservation of the structure and function of biological molecules is of fundamental importance to biology, biochemistry, and medicine. Biological materials, such as proteins, enzymes, cells, tissues, nucleic acid, semen, blood and its components, mammalian organs, and foodstuffs must often be stored and preserved for later use. Preservation of these biological materials is usually achieved by either freezing or drying, or a combination of the two processes. There are several commonly-used drying techniques: drying by evaporation into a moving gas stream (ambient air-drying), drying under vacuum at ambient temperatures (vacuum-drying), or drying by contacting a fine mist of droplets with warm air (spray-drying). Simple freezing is often done when drying is either harmful or unnecessary. Certain biological materials are best preserved by freeze-drying (lyophilization), a two-step process in which the sample is first frozen and then dried at low temperature under vacuum.
The structure and function of most biological materials is dependent upon their aqueous environment. Therefore, changes to their aqueous environment resulting from freezing and drying processes can often have drastic consequences for a biological material. Furthermore, freeze-drying combines the stresses due to both freezing and drying. The freezing step of this process can have undesirable side effects, such as the denaturation of proteins and enzymes, and rupture of cells. These effects result from mechanical, chemical, and osmotic stresses induced by crystallization of ice in these materials. As a result, the activity of the biological material upon rehydration is lost either in its entirety, or to such a significant extent that the material is no longer useful for its intended purpose.
To prevent or reduce the adverse effects upon reconstitution or rehydration, protective agents, such as cryoprotectants or lyoprotectants (freeze-drying) are used. For such protective agents to be effective, they must be non-toxic to the biological material at the concentrations encountered during the preservation process, and must interact favorably with water and with the biological material. Various protective agents have been used in the art, with varying degrees of success. These include fish proteins, certain polymers, skim milk, glycerol, dimethyl sulfoxide, and disaccharides, such as trehalose. Unfortunately, suitable protective agents and cryopreservation protocols have been developed only for a limited number of systems.
Disaccharides, such as sucrose and trehalose, are natural cryoprotectants. Trehalose is a particularly attractive cryoprotectant because it has actually been isolated from plants and animals that remain in a state of suspended animation during periods of drought. Trehalose has been shown to be an effective protectant for a variety of biological materials, both in ambient air-drying and freeze-drying. Research has shown, (see Crowe, J. H., Crowe., L. M., and Mouriadian, R., Cryobiology, 20, 346-356 (1983)), that liposomes dried in the presence of trehalose retain both their functional and structural integrity upon rehydration. U.S. Pat. No. 5,556,771 discloses the use of trehalose, or trehalose in combination with polyvinylpyrrolidone to preserve reverse transcriptase and RNA polymerase. U.S. Pat. No. 5,512,547 discloses the use of trehalose to preserve botulinum neurotoxin. Likewise, U.S. Pat. No. 4,891,319 discloses a method of protecting proteins and other biological macromolecules, such as enzymes, serum, serum complement, antibodies, antigens, fluorescent proteins and vaccine components using trehalose. Specifically, an aqueous mixture containing the macromolecule and trehalose is dried at a temperature above freezing in the presence of 0.05 to about 20% trehalose by weight of the aqueous system.
However, there are some drawbacks associated with the use of trehalose as the sole cryoprotectant. To preserve many biological materials by freeze-drying, large amounts of trehalose must be used; concentrations of trehalose greater than 60% by weight of a given preservation medium are sometimes necessary. This is costly. Further, a high concentration of trehalose reduces the solubility of other solutes in the system.
Thus, it has been proposed to use trehalose in combination with a polymeric gelling agent, such as carboxymethylcellulose or carboxyethylcellulose. It has been suggested for human blood that saccharides combined with polymers are even more effective cryoprotectants than pure trehalose. See U.S. Pat. No. 5,171,661; Sutton, R. L., J. Chem. Soc. Faraday Trans., 87, 3747 (1991). Unfortunately, attempts to confirm the beneficial effect of the gelling agents have been unsuccessful. (G. Spieles, I. Heschel, and G. Rau, CryoLetters 17, 43-52 (1996), J. H. Crowe, A. E. Oliver, F. A. Hoekstra, and L. M. Crowe, Cryobiology 35, 20-30 (1997).). Moreover, this protective combination cannot be used for medical purposes, because the polymer gelling agents are not accepted well by the human body. As a result, this combination is not very useful, and does not provide much, if any, practical improvement over the use of trehalose alone.
Another, more serious problem associated with the use of trehalose is that biological materials preserved using trehalose alone are not storage stable for extended periods of time, especially those stored at superambient temperatures and/or in humid environments. In other words, biological materials preserved with trehalose can lose their activity in a matter of hours or days, depending on the humidity and temperature of the storage conditions.
Therefore, at present, freeze-drying with trehalose is of limited use for extended term storage of biological materials, such as proteins, enzymes, cells, tissues, nucleic acid, semen, blood and its components, mammalian organs, and foodstuffs, over a wide range of storage conditions, because the material will degrade, and will not have sufficient activity upon reconstitution. From a practical standpoint, this is clearly unacceptable for medical products, as one of the reasons for preserving the materials in the first place is to provide a storage-stable product.
Nor can many of the various room temperature drying techniques be effectively used at present. These methods, while less complicated and less costly than freeze-drying, are generally more destructive to biological materials. Many biological materials are more prone to gross conformational changes and unwanted reactions when preserved using methods that take place at ambient temperature than when freeze-drying is used. As a result, even where presently known protective agents are used, the activity of many rehydrated biological materials is both unsatisfactory in its own right, and significantly less than if preserved by freeze-drying.
Thus, a need exists for a protectant mixture that is useful for a wide range biological materials. A further need exists for a protectant mixture that can be effectively used in both freeze-drying processes and drying processes involving ambient-temperature drying. There is also a need for a protectant mixture that is less costly than those presently being used. Finally, and very importantly, there is a need for a protectant mixture that provides stable media for preservation of biological materials over extended periods of time at elevated temperatures and varying degrees of humidity, which can be encountered during shipping and storage of materials, while still retaining a significant amount of activity upon rehydration.
All of these needs are met by the protectant mixture, aqueous protective medium and resulting preserved biological material compositions of the present invention.
It has been found that a protectant mixture for use in preserving biological materials, comprising: (a) at least one polyhydroxy compound, where the total amount of polyhydroxy compound in the mixture is from about 5% to about 60% by weight of the mixture if the mixture is an aqueous solution and from about 10% to about 95% by weight if the mixture is in solid form; and (b) phosphate ions, where the total amount of phosphate ions in the medium is such that the molar ratio of phosphate ions to hydroxyl groups in the polyhydroxy compound is from about 0.025 to about 0.625, can be used with a wide variety of biological materials to provide an aqueous preservation medium. This aqueous preservation medium can then be used in a multiplicity of preservation processes, including freezing, freeze-drying and other drying processes, such as spray-drying, vacuum-drying, or ambient air-drying, to provide a stable, preserved composition of the biological material of interest. This preserved composition is stable for extended periods of time at superambient temperatures and/or relative humidity. Further, when the preserved biological material composition is rehydrated, the structural and functional integrity of the preserved biological material has been retained to such an extent that the biological material can be used for its intended purpose.
Therefore, the present invention also provides a method for preparing a preserved biological material composition from the above-noted preservation medium, as well as the preserved biological material composition itself.